Detection of pathogenic Aeromonas spp. from a simulated water distribution system using PCR

Recently the EPA placed Aeromonas hydrophila on the Candidate Contaminant List (CCL). It has long been known to be a pathogen of cold blooded animals and now is a suspected human opportunistic pathogen as well. Among the various virulence factors produced by A. hydrophila, the cytolytic enterotoxin (AHCYTOEN) is by far one of the most important contributors to the pathogenicity of the organism. This factor is also produced by other pathogenic Aeromonas spp. In this study, PCR technology was used to detect AHCYTOEN gene from a simulated water distribution system. A set of primers was selected to amplify the unique sequence of a pathogenic island, AHCYTOEN gene. To examine the sensitivity of the PCR, serial dilutions of pure A. hydrophila culture were tested. The PCR technique used was sensitive enough to detect samples containing less than 10.0 cells/ml. Source water, bulk water, and simulated distribution biofilm samples were examined for the gene. Biofilm and bulk water samples exposed to raw source water were collected on 4 occasions during a 24-day period. PCR technology detected the AHCYTOEN gene from 100 % of the bulk water samples and 85% of tightly bound biofilm (TB) samples from a simulated water distribution system while no positive results were observed in loosely bound biofilm samples (LB). After the inlet line of the system was changed to normally treated distribution water, 11 biofilm samples were collected on 3 occasions during 15 day sampling period along with bulk water samples. No positive results were observed from the bulk water and LB samples while 91% of TB samples tested for the presence of the gene. No significant difference was observed in detection by PCR from biofilm samples before and after the switch to chloraminated water.Department of BiologyThesis (M.S.